Methods
Subjects . COVID-19 patient blood samples used for
immunophenotyping were obtained during patients’ visit or
hospitalizations at SUNY Downstate Medical Center in New York from May
through to July 2020. Patients were defined as 1) non-hospitalized, with
and without presentation of COVID-19 symptoms and 2) hospitalized with
presentation of COVID-19 symptoms. Peripheral blood from venipuncture
was drawn into EDTA and Heparin coated vacutainer tubes for
immunophenotyping and processed within 48h of blood draw. Control blood
samples from healthy volunteers without SARS-CoV-2 infection or
co-morbid conditions were collected after obtaining written informed
consent.
Flow Cytometry . Whole blood was stained per the clinical standard
immunophenotyping protocols (Amerimmune LLC, Fairfax, VA). The samples
were stained with the multiple antibody combinations for 30 minutes at
4˚C. RBCs were lysed using BD FACS lysis solution (BD Bioscience, San
Jose, CA) as per manufacture directions. In brief, freshly obtain
peripheral blood mononuclear cells (PBMC) were separated from 2 mL of
whole blood within 24h of collection and diluted 1:1 with phosphate
buffered saline pH 7.2 (PBS) (Thermo Fisher Scientific, Carlsbad, CA)
using Lymphoprep (Stem cell Technologies, Cambridge, MA) and Accuspin
tubes (Sigma-Aldrich, St. Louis, MO) as per manufactures directions.
PBMCs’ were washed in PBS and resuspended in 0.5 mL PBS. 100 µL of the
PBMCs were immunostained with a mixture of antibodies at 4℃ for 1 hour.
Cells were washed and resuspended in PBS prior to acquisition.
Antibodies used for the immune phenotyping of patient samples and gating
strategy for active caspase-1 staining are detailed in the Supplementary
information section of the manuscript ( Supplemental Table 1).
Apoptosis (caspase 3/7) and pyroptosis (caspase-1) were measured by flow
cytometry using fluorescent-labeled inhibitors of caspase probe assay
(FLICA; Immunochemistry Technologies, Minneapolis, MN). As a control,
PBMCs were stimulated with nigericin for 2 hours. FAM-FLICA probes
specific for caspase-1 or caspase 3/7 were added to 50 µl PBMC or whole
blood and incubated for 1h at 37℃. Cells were subsequently washed and
stained with a cocktail of fluorescently conjugated antibodies against
CD45 PE-CY7 [HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4],
CD45RO PerCP-EF710 [UCHL1] and Viability Dye 780 (Thermo Fisher
Scientific, Carlsbad, CA). Red blood cells were identified as
CD235-positive cells in the experiments where caspase3/7 activity was
measured. Samples were acquired on a 3 laser BD FACS Canto 10. CS&T
beads (BD Bioscience, San Jose, CA) were acquired daily to ensure
consistent performance of the BD FACS Canto 10 Canto10. The BD FACS
Canto 10 utilized for this study has been validated for T, B, NK and
Dendritic cell immunophenotyping clinical diagnostic testing. Denovo FCS
Express v6 clinical edition (De Novo Software, Pasadena, CA) was used
for flow cytometric analyses.
Plasma experiments . Plasma was separated from whole blood
following centrifugation at 960 RCF. Cells (RBC and WBC) were either
incubated at 37 °C alone or in the presence of trypsin for 1 hour then
washed with 10 packed cell volumes of RPMI 1640 incomplete medium.
Plasma was either held at room temperature (18 - 25°C) or heat
inactivated at 56°C for 1 hr. Plasma was added back to the RBC/WBC in a
1:1 ratio and incubated overnight, rocking at room temperature. RBC
caspase 3/7 activity was measured as described under flow cytometry.
Public SARS-CoV-2 and COVID-19 Transcriptome Analyses . Single
cell RNA-Seq data from three COVID-19 participants that were ventilated
and diagnosed with acute respiratory distress syndrome at 2-16 days
after symptom onset and from 6 healthy controls was accessed from GEO
(30). RNA-Seq data from cell lines infected in vitrowith SARS-CoV-2 was accessed from GEO: GSE147507 (31).Expression values for Caspase genes were normalized by DESeq2.
Ex vivo stimulation studies . Active caspase-1 in COVID-19
infected patient samples. Whole blood from a COVID-19 positive patient
was either (A) untreated or (B) treated with the pan-caspase inhibitor
emricasan (Sigma Aldrich, MO, SML2227-5MG) or selective caspase-1
inhibitor VX765 overnight at 37 degrees in a water bath. Subsequently,
PBMCs were purified (Accuspin System – Histopaque 1077; Sigma Aldrich,
MO, A6929) and incubated with nigericin (Immunochemistry Technologies,
MN) for 2h. A Fam-FLICA probe specific for active caspase-1 was added
to 50 µl PBMC, incubated for 1h at 37℃. PMBCs were washed with cell
wash buffer (Immunochemistry Technologies, MN) and stained with a
cocktail of fluorescently conjugated antibodies against CD45 PE-CY7
[HI30], CD3 AF700 [UCHT1], CD4 PE [RPA-T4], CD45RO
PerCP-EF710 [UCHL1] and Viability Dye 780 (Thermo Fisher Scientific,
Carlsbad, CA). Lymphocytes were identified using a standard gating
schematic which incorporated gating of lymphocytes on an FSC/SSC plot
and singlets on a FSC-A/FSC-H plot. Lymphocytes were further identified
as CD45+ on a CD45/SSC plot and subsequent CD3-, CD3+ and CD3+CD4+ cells
were identified on a CD45+ CD3/CD4 plot.
Statistical analysis . Demographic and HIV-related characteristics
were described using the median, first quartile (Q1), and third quartile
(Q3) for continuous variables and frequency for categorical variables.
Differences among continuous variables were evaluated by either the
Mann-Whitney, student t test or Krustal-Wallis test with Dunn’s
multiple comparisons. Relationships among parameters were examined by
Pearson correlation for continuous variables. All statistical tests were
performed with GraphPad Prism version 8.0 (Graphpad Software Inc., CA,
USA). Statistical significance is indicated as *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001.
P-values ≤0.100, but not significant, are noted as statistical trends.
Study approval . All clinical investigations were conducted
according to Declaration of Helsinki principles. All human studies were
approved by institutional review boards (IRB 269846-10 and 1285028
protocols from State University of New York Downstate Medical Center and
Amerimmune respectively). Written informed consent was received from
participants prior to inclusion in the study.